Structural proteomics

1
Structure Determination and Analysis X-ray
Crystallography
2
Source
Sample
Detector
Intensity of diffracted beam
Diffraction
X-rays
3
Energy hc ?
4
X-rays

• Unlike using a light microscope, there is no way
  of re-focusing diffracted x-rays.
• Instead we must collect a diffraction pattern
  (spots).

• It is possible to translate information in the
  diffraction pattern into atomic structure using
  Braggs law, which predicts the angle of
  reflection of any diffracted beam from specific
  atomic planes

5
A typical crystallography experiment
Pure protein Grow crystal Characterize
crystals Collect diffraction data Solve phase
problem Calculate electron density
map Build/rebuild model Refine model Analyze
structure
6
The Beginning
7
Principles of X-ray diffraction
What is a crystal?

• The unit cell is the basic building block of the
  crystal
• The unit cell can contain multiple copies of the
  same molecule whose positions are governed by
  symmetry rules

8
Proteins and crystallisation

• What type of protein is it? Has anything similar
  been crystallized before?
• Proteins must be pure (gt 99) fully folded
  Check the activity of your protein if you have an
  assay Check folding by other spectroscopic
  methods

• Proteins must be homogenous monodispersed.
• Need large amount (mg quantities)
• Is it stable ( salt, pH, temp)
• Will modifications have to be made?

9

• Crystallisation of proteins controlled
  precipitation of the protein.
• Protein aggregates associate form
  intermolecular contacts that resemble those
  found in the final crystal. Aggregates reach the
  critical nuclear size, growth proceeds by
  addition of molecules to the crystalline lattice.
  
• The processes of nucleation and crystal growth
  both occur in supersaturated solutions.

• Process controlled by
• Temp
• pH
• Salt conc
• Precipitants (PEG, ethanol)

10
Diffraction Apparatus
11
Synchrotron radiation
More intense X-rays at shorter wavelengths mean
higher resolution much quicker data collection
12
Experimental setup
13
Mounting crystals
Remove cover slip and fish out crystal with a
small nylon loop
Surface tension of the liquid in the loop holds
crystal in place
Mount loop on goniostat in a stream of nitrogen
gas
14
Diffraction

• Each image represents the rotation of the crystal
  1 degree in the X-ray beam.
• Each images gives us the position of each spot
  relative to all the others there intensity.
• Intensity square of amplitude.

15
Diffraction Principles
nl 2dsinq
16
Diffraction Principles
Corresponding Diffraction Pattern
A string of atoms
17
The reciprocal lattice and the geometry of
diffraction
18
Spacing between diffraction spots defines unit
cell
1/b
1/a
19
Waves the phase problem
The amplitudes of the diffracted X-rays can be
experimentally measured, but the phases cannot
phase problem.
i.e. we dont know the phase of each diffracted
ray relative to the others!
20
The Phase Problem

• Diffraction data only records intensity, not
  phase information (half the information is
  missing)
• To reconstruct the image properly you need to
  have the phases (even approx.)
• molecular replacement
• direct methods
• isomorphous replacement
• anomolous dispersion

21
Structure factors Fourier transforms
unit cell F (h,k,l) V?x0 ?y0 ?z0
?(x,y,z).exp2?I(hx ky lz).dxdydz A
reflection electron density All reflections
phase ?(x,y,z) 1/V ?h?k?l ?F
(h,k,l)?exp2?I(hx ky lz)
i?(h,k,l) Electron density amplitude At a
point 

• The vector (amplitude and phase) representing the
  overall scattering from a particular set of Bragg
  planes is termed the structure factor (F).
• Structure factors for various points on the
  crystal lattice correspond to the Fourier
  transform of electron density within the unit
  cell and vice-versa.

22
Fourier Transform of a molecule
F T
23
Fourier Transform of a crystal
24
The Phase Problem

• Diffraction data only records intensity, not
  phase information (half the information is
  missing)
• To reconstruct the image properly you need to
  have the phases (even approx.)
• molecular replacement
• direct methods
• isomorphous replacement
• anomolous dispersion

25
Molecular replacement

• Requires a starting model for structure
• Can calculate back from structure to electron
  density to structure factors
• Works if model is 30 to 40 identical to correct
  answer

26
Molecular Replacement
By determining the correct orientation and
position of a molecule in the unit cell using a
previously solved structure as a search model.
This model can then be used to calculate phases
27
Isomorphous replacement (IR)

• Provides indirect estimates of the protein phase
  angles by observing the interference effects of
  the intensities on scattered beams by a heavy
  atom marker.
• All the electrons in the heavy atom will scatter
  essentially in the same phase.
• We can solve the positions of these heavy atoms
  because they are few in number and strong in
  signal.
• Using this estimate we can deduce the positions
  of the protein atoms and their phases

28
Anomalous scattering

• Scattering information of an atom whose
  absorption frequency is close to the wavelength
  of the source beam produces phase information
• Resolved anomalous scattering requires intensity
  measurements at one wavelength
• Multi-wavelength anomalous dispersion, requires
  intensity measurements at several wavelengths

29

• Using the structure factor calculation we can
  produce electron density maps for the whole
  protein.
• We then fit our protein model (co-ordinates
  X,Y,Z) inside the map.

30
Resolution
1.2 Å
2 Å
3 Å
31
Resolution
6Å Outline of the model, feature such as helices
can be identified. 3Å Can trace polypeptide
chain using sequence data, establish folding
topology. Assign side chains. 2Å Accurately
establish mainchain conformation, assign
sidechains without sequence data, I.d water
molecules. 1.5Å Individual atoms are almost
resolved, detailed discription of water
structure. 1.2Å Hydrogen atoms may become
visible.
32
Final Structure
But the work is not over yet!
33
Refinement

• The process of building and rebuilding a model
  can cause many errors in the structure.
• Bond length,
• Bond angle
• Atomic clashes etc
• It is necessary to subject the structure to
  refinement in order to remove these errors and
  produce a better structure.
• Minimization
• Thermal parameters
• In order to further improve the model, it is
  refined using a simulated annealing protocol
• Refinement progress is monitored by following the
  agreement between the the observed data ( data
  collected) and the calculated data (data
  calculated from current model) R factor

34
Quality of the structure?

• R-factor The agreement between the the observed
  data (data collected) and the calculated data
  (data calculated from current model) the lower
  the number the better typically around 20
• Resolution The higher the resolution the more
  detail that can be seen 3.0Å is fairly low whilst
  1.1Å is approaching atomic resolution
• B-factor Measure of thermal motion. i.e. how much
  energy each atom contains. Gives us information
  on mobility stability
• Rms deviation Deviation of bond lengths angles
  from ideal

35
Rms deviation of bond length bond angle
Deviation of bond lengths angles from ideal.
All based on the geometry of small molecules. Rms
deviation for bond lengths should be less than
0.02Å and less than 4º for bond angles
Determined using a Ramachandran plot.
36
Absorption of Light
37
Absorption in the UV and visible range

• Protein chromophores
• Peptide bond
• Amino acid side chains
• Prosthetic groups

• Amino acid side chain absorbance
• Asp, Glu, Asn, Gln, His and Arg have transitions
  at the same wavelength where peptide absorbs

• Peptide bond absorbance
• 210 nm due to n ? ?? transition
• 190 nm due to ? ? ?? transition

Protein concentration can be measured by
measuring absorbance at 280 nm and by assuming
that 1 mg ml-1 solution of protein has absorbance
of 1.0
38
Absorption and emission spectra of individual
tryptophan residues, in the absence of energy
transfer
39
Fourth derivative absorption spectrum

• Fourth derivatives of the absorption spectra have
  been documented as a valuable tool for studying
  structural changes in proteins.
• Protein fourth derivative spectra have been shown
  to be very sensitive to changes in the
  microenvironment (polarity, hydration,
  hydrophobic interactions, packing density) of
  tyrosine and tryptophan residues

Chauhan and Mande, Biochem J, 2001
40
Measurements of conformational properties using
optical activity
41
Linearly polarised light
Right circular polarisation
Left circular polarisation
42

• Nearly all molecules of life are optically active
• There are four ways that an optically active
  sample can alter the properties of transmitted
  light optical rotation, ellipticity, circular
  dichroism, circular birefringence

Linear
Elliptical
Circular
43

• After passing through an optically active
  absorbing sample, the light is changed in two
  aspects
• The maximal amplitude E is no longer confined to
  a place, instead it traces an ellipse
• Ellipticity tan-1 (minor/major axis)
• The orientation of the ellipse is an indication
  of optical activity. If the sample did not
  absorb any light, the ellipse would such small
  axial ratio that it would be equivalent to a
  plane-polarised light. In this case we will say
  that the plane polarised light has been rotated.
• Orientation of the ellipse is the optical
  rotation. Optical rotation as a function of
  wavelength is called the optical rotatory
  dispersion (ORD).

44
Circular Dichroism
45
CD spectrum of a protein
46
(No Transcript)
47
Where can Circular Dichroism be used?
48
Measurements of conformational properties using
fluorescence
49
Fluorescence

• Chromophores are components of molecules which
  absorb light
• They are generally aromatic rings

50
Fluorescence
Jablonski Diagram
Singlet States
Triplet States
Vibrational energy levels
S2
Rotational energy levels
Electronic energy levels
T2
S1
IsC
ENERGY
T1
ABS
FL
I.C.
PH
IsC
S0
Vibrational sublevels
ABS - Absorbance S 0.1.2 - Singlet Electronic
Energy Levels FL - Fluorescence T 1,2 -
Corresponding Triplet States I.C.- Nonradiative
Internal Conversion IsC - Intersystem
Crossing PH - Phosphorescence
51
Simplified Jablonski Diagram
52
Fluorescence
The longer the wavelength the lower the energy
The shorter the wavelength the higher the
energy eg. UV light from sun causes the sunburn
not the red visible light
53
Fluorescence Excitation Spectra
Intensity related to the probability of the
event
Wavelength the energy of the light absorbed or
emitted
54
Corrected excitation spectra (corrected for
source output and monochromator throughput) can
be obtained by using a reference channel equipped
with a "quantum counter". This is a concentrated
dye solution (typically 3 mg/mL rhodamine B in
ethylene glycol). A tiny fraction of the
excitation beam is diverted to the reference
detector. The quantum counter absorbs all of this
light, and converts it (with 100 efficiency to
fluorescence), the intensity of which is
independent of wavelength between 220 and 580 nm.
Any changes in lamp output or monochromator
throughput will cause corresponding alterations
in the output of the reference channel. By
dividing the fluorescence signal by the reference
signal, these wavelength-dependent variations are
cancelled out. Unfortunately, the quantum
counter will not entirely correct the emission
spectrum. However, instrument manufacturers
supply correction factors for their
monochromators. Application of these will give an
approximately correct spectrum. If more accuracy
is needed, the spectrum of a known standard
compound (fluorescing in the region of interest)
can be compared to published standards.
j. Biological fluorophores 1) Intrinsic
fluorophores a) Proteins Tryptophan
dominates protein fluorescence spectra
- high molar absorptivity - moderate
quantum yield - ability to quench tyrosine
and phenylalanine emission by
energy transfer.
Free tyrosine has a relatively high fluorescent
output, but is strongly quenched by trptophan in
native proteins. Unless tyrosine and tryptophan
are absent, emission from phenylalanine is not
observed in protein fluorescent spectra.
55
Tryptophan is a good fluorophore
note that this fluorescence expt used an
excitation l of 270nm
note that the fluorescence looks like a mirror
image of the 280nm absorption peak (and not the
220nm peak)

• we can consider solvent effects on its emission
  wavelength in the same way we did for
  absorption...

56
Absorption vs Emission for Trp

• comparing our diagrams for absorption and
  emission and assuming that protein interiors
  behave like organic solvents(!) we predict

in water
buried in protein
57
Effect of Ca2 on Intrinsic Trp-fluorescence and
on Fluorescence Anisotropy
Blue shift and intensity enhancement upon
addition of Ca2
? Wild type Dome loop mutant
Change in anisotropy upon titration in the wild
type, but not in the mutant
58
Raman Scatter

• A molecule may undergo a vibrational transition
  (not an electronic shift) at exactly the same
  time as scattering occurs
• This results in a photon emission of a photon
  differing in energy from the energy of the
  incident photon by the amount of the above energy
  - this is Raman scattering.
• The dominant effect in flow cytometry is the
  stretch of the O-H bonds of water. At 488 nm
  excitation this would give emission at 575-595 nm

59
Rayleigh Scatter

• Molecules and very small particles do not absorb,
  but scatter light in the visible region (same
  freq as excitation)
• Rayleigh scattering is directly proportional to
  the electric dipole and inversely proportional to
  the 4th power of the wavelength of the incident
  light

the sky looks blue because the gas molecules
scatter more light at shorter (blue) rather than
longer wavelengths (red)
60
Probes for Proteins
Probe Excitation Emission

• FITC 488 525
• PE 488 575
• APC 630 650
• PerCP 488 680
• Cascade Blue 360 450
• Coumerin-phalloidin 350 450
• Texas Red 610 630
• Tetramethylrhodamine-amines 550 575
• CY3 (indotrimethinecyanines) 540 575
• CY5 (indopentamethinecyanines) 640 670

61
Probes for Nucleic Acids

• Hoechst 33342 (AT rich) (uv) 346 460
• DAPI (uv) 359 461
• POPO-1 434 456
• YOYO-1 491 509
• Acridine Orange (RNA) 460 650
• Acridine Orange (DNA) 502 536
• Thiazole Orange (vis) 509 525
• TOTO-1 514 533
• Ethidium Bromide 526 604
• PI (uv/vis) 536 620
• 7-Aminoactinomycin D (7AAD) 555 655

62
DNA Probes

• AO
• Metachromatic dye
• concentration dependent emission
• double stranded NA - Green
• single stranded NA - Red
• AT/GC binding dyes
• AT rich DAPI, Hoechst, quinacrine
• GC rich antibiotics bleomycin, chromamycin A3,
  mithramycin, olivomycin, rhodamine 800

63
Probes for Ions

• INDO-1 Ex350 Em405/480
• QUIN-2 Ex350 Em490
• Fluo-3 Ex488 Em525
• Fura -2 Ex330/360 Em510

64
pH Sensitive Indicators
Probe Excitation Emission

• SNARF-1 488 575
• BCECF 488 525/620
• 440/488 525

2,7-bis-(carboxyethyl)-5,6-carboxyfluorescein
65
Probes for Oxidation States
Probe Oxidant Excitation Emission

• DCFH-DA (H2O2) 488 525
• HE (O2-) 488 590
• DHR 123 (H2O2) 488 525

DCFH-DA - dichlorofluorescin diacetate HE -
hydroethidine DHR-123 - dihydrorhodamine 123
66
Specific Organelle Probes
Probe Site Excitation Emission

• BODIPY Golgi 505 511
• NBD Golgi 488 525
• DPH Lipid 350 420
• TMA-DPH Lipid 350 420
• Rhodamine 123 Mitochondria 488 525
• DiO Lipid 488 500
• diI-Cn-(5) Lipid 550 565
• diO-Cn-(3) Lipid 488 500

BODIPY - borate-dipyrromethene complexes NBD -
nitrobenzoxadiazole DPH - diphenylhexatriene TMA
- trimethylammonium
67
Other Probes of Interest

• GFP - Green Fluorescent Protein
• GFP is from the chemiluminescent jellyfish
  Aequorea victoria
• excitation maxima at 395 and 470 nm (quantum
  efficiency is 0.8) Peak emission at 509 nm
• contains a p-hydroxybenzylidene-imidazolone
  chromophore generated by oxidation of the
  Ser-Tyr-Gly at positions 65-67 of the primary
  sequence
• Major application is as a reporter gene for assay
  of promoter activity
• requires no added substrates

68
Energy transfer
transfer
emission
excitation
phycoerythrin-Texas Red ECD phycoerythrin-cyanine5
PC5
69
Energy Transfer

• Effective between 10-100 Å only
• Emission and excitation spectrum must
  significantly overlap
• Donor transfers non-radiatively to the acceptor

Molecule 1
Molecule 2
Fluorescence
Fluorescence
ACCEPTOR
DONOR
Intensity
Absorbance
Absorbance
Wavelength
70
Energy transfer
excited states
ground state
A
B